The invention relates generally to the field of drug screening assays. More particularly, the invention relates to methods and compositions for identifying molecules that modulate expression of true tissue morphogenic proteins.
A class of proteins recently has been identified, the members of which are true tissue morphogenic proteins. The members of this class of proteins are characterized as competent for inducing the developmental cascade of cellular and molecular events that culminate in the formation of new organ-specific tissue, including any vascular and connective tissue formation, as required by the naturally occurring tissue. Specifically, the morphogens are competent for inducing all of the following biological functions in a morphogenically permissive environment: (1) stimulating proliferation of progenitor cells; (2) stimulating differentiation of progenitor cells; (3) stimulating the proliferation of differentiated cells and (4) supporting the growth and maintenance of differentiated cells. For example, the morphogenic proteins can induce the full developmental cascade of bone tissue morphogenesis, including the migration and proliferation of mesenchymal cells, proliferation and differentiation of chondrocytes, cartilage matrix formation and calcification, vascular invasion, osteoblast proliferation, bone formation, bone remodeling, and hematopoietic bone marrow differentiation. These proteins also have been shown to induce true tissue morphogenesis of non-chondrogenic tissue, including dentin, liver, and nerve tissue.
A particularly useful tissue morphogenic protein is human OP-1 (Osteogenic Protein-1), described in U.S. Pat. No. 5,011,691; U.S. Pat. No. 5,266,683 and Ozkaynak et al. (1990) EMBO J. 9: 2085-2093. Species homologues identified to date include, but are not limited to, mouse OP-1 (see U.S. Pat. No. 5,266,683) and the Drosophila homologue 60A, described in Wharton et al. (1991) Proc. Nat. Acad. Sci. USA 88:9214-9218). Other closely related proteins include OP-2 (Ozkaynak (1992) J. Biol. Chem. 2:25220-25227 and U.S. Pat. No. 5,266,683); BMP5, BMP6 (Celeste et al. (1991) Proc. Natl. Acad. Sci. 8:9843-9847) and Vgr-1 (Lyons et al. (1989). These disclosures are incorporated herein by reference.
It previously has been contemplated that these tissue morphogens can be administered to an animal to regenerate lost or damaged tissue. Certain complications, however, presently are encountered during the production, formulation and use in vivo of therapeutic macromolecules, such as morphogen proteins. For example, such proteins are typically produced by fermentation or culture of suitable host cells. Any biological product produced from such host cells for use in humans presently must be shown to be essentially free of host cell contaminants, such as secreted or shed proteins, viral particles or degradation products thereof. Providing such assurance can add significantly to the cost and technical difficulty of commercial production of biological macromolecules. Furthermore, appropriate formulations must be developed for conferring commercially reasonable shelf life on the produced macromolecule, without significant loss of biological efficacy. An additional complicating factor arises when circumstances warrant an extended course of therapeutic treatment with the produced and formulated macromolecule: the treated mammal may develop an immunological response to the macromolecule, and any such response may interfere with effectiveness thereof. In extreme circumstances, treatment must be discontinued.
Alternatively, administering a molecule capable of modulating expression of the endogenous tissue morphogen is an effective means for providing morphogen to a site in vivo. For example, DNA sequences have been identified in the OP-1 gene promoter that resemble wt-1/Egr-1 consensus sequences, TLC binding sequences, FTZ binding sequences and steroid binding sequences (see WO 95/33831, the disclosure of which is incorporated herein by reference). Thus, molecules to which these regulatory sequences are responsive are likely modulators of the OP-1 gene and can influence its expression.
It is an object of this invention to provide compositions and methods of identifying compounds which can modulate expression of an endogenous tissue morphogen, particularly OP-1 and other members of the larger genus of true tissue morphogens. The compounds thus identified have utility both in vitro and in vivo. Useful compounds contemplated include at least those that are capable of stimulating transcription and/or translation of the OP-1 gene, as well as compounds capable of inhibiting transcription and/or translation of the OP-1 gene, via OP-1 non-coding DNA sequences resembling consensus sequences for Pax homeobox genes, in particular, the Pax 6 or Pax 2 genes.
These and other objects and features of the invention will be apparent from the description, drawings and claims which follow.
The invention features compositions and methods for screening candidate compounds for their ability to modulate the effective local or systemic levels of endogenous morphogen, particularly OP-1, in an organism. In one aspect, the method is practiced by: (I) incubating one or more candidate compounds with cells transfected with a DNA sequence encoding at least a portion of a morphogen non-coding DNA sequence that is responsive to a Pax homeobox gene and which is competent to act on and affect expression of a reporter gene with which it is operatively associated; (2) measuring the level of reporter gene expression in the transfected cell, and (3) comparing the level of reporter gene expressed in the presence of the candidate compound with the level of reporter gene expressed in the absence of the candidate compound. The level of an expressed reporter gene product in a given cell culture, or a change in that level resulting from exposure to one or more compound(s) indicates that the compound can also modulate the level of the morphogen normally associated with the non-coding sequence. Specifically, an increase in the level of reporter gene expression is indicative of a candidate compound""s ability also to increase morphogen expression in vivo. Similarly, a decrease in the level of reporter gene expression is indicative of a candidate compound""s ability also to decrease or otherwise interfere with morphogen expression in vivo. The above method is particularly useful for identifying compounds that are capable of influencing Pax mediated OP-1 gene expression.
The methods of the invention can therefore be used to identify compounds showing promise as therapeutics for various in vivo and ex vivo mammalian applications, as well as to identify compounds having numerous utilities. For example, compounds that modulate morphogen expression by stimulating Pax 2 or Pax 6 mediated transcription of a morphogen can be used in vivo to correct or alleviate a disease condition, to regenerate lost or damaged tissue, to induce cell proliferation and differentiation, and/or to maintain cell and tissue viability and/or a differentiated phenotype in vivo or ex vivo. The compounds also can be used to maintain the viability of, and the differentiated phenotype of, cells in culture. The various in vivo, ex vivo, and in vitro utilities and applications of the morphogenic proteins described herein are well documented in the art. See, for example, US 92/01968 (WO 94/03200), filed Mar. 11, 1992; US 92/07358 (WO 93/04692), filed August 28; PCT US 92/0743 (WO 93/05751), filed Aug. 28, 1992; US 93/07321 (WO 94/03200), filed Jul. 29, 1993; US 93/08808 (WO 94/06449), filed Sep. 16, 1993; US93/08885 (WO94/06420), filed Sep. 15, 1993, and U.S. Pat. No. 5,266,683.
In another aspect, the invention further provides vectors and cells useful for morphogen, particularly OP-1, therapy. In one embodiment, the invention features a vector having a reporter gene operatively associated with at least a portion of one or more OP-1 non-coding sequences responsive to Pax homeobox gene products. The OP-1 non-coding sequences comprise at least a first Pax responsive OP-1 modulating element which is responsive to a first Pax gene expression product. In other embodiments, vectors further comprise a second non-coding sequence comprising at least a second Pax-responsive OP-1 modulating element which is responsive to a second Pax gene expression product; in such embodiments, the first and second Pax gene expression product differ. OP-1 non-coding sequences which are Pax responsive OP-1 modulating elements can be selected from nucleotides 1-3317 of SEQ ID No. 1. Also anticipated to be similarly useful are certain of the non-coding sequences of other species homologues of OP-1 and proteins closely related to OP-1. For example, other non-coding DNA that is responsive to Pax gene products or homologues thereof can be used to identify modulators of specific morphogens, or other factors capable of modulating morphogen gene expression.
In another embodiment, the vector can include a non-coding OP-1-specific sequence selected from at least one of the following sequence segments of SEQ. ID No. 1 presented below, which defines approximately 3.3 Kb of 5xe2x80x2 non-coding human genomic OP-1 sequence. Preferred vectors comprise sequence segments including nucleotides 1-3317, as well as shorter fragments of this region of DNA such as approximately nucleotides 108-121, 139-154, 157-167, 365-378, 491-503, 598-613, 737-747, 891-903, 994-1006, 1123-1140, 1144-1161, 1285-1297, 1750-1762, 2001-2023, 2365-2378, 2931-2944 of SEQ. ID No. 1, including allelic, species and other sequence variants thereof. As base 2790 is the mRNA start site, other preferred sequences include approximately 2790-3317, representing transcribed but not translated 5xe2x80x2 non-coding sequence and shorter fragments of this DNA region. Other preferred regions of the 5xe2x80x2 non-coding region of SEQ. ID No. 1 include regions comprising a cluster of several Pax responsive elements, such as, for example, approximately 1-2073, 1-1297, 1-2691, 1-378, 491-1006, 1750-2023, 1750-2378, 1750-2691, 1750-2944. In certain embodiments, non-coding sequences correspond to part or all of SEQ. ID No. 2 and/or SEQ. ID No. 3, including allelic, species and other sequence variants thereof. In yet other embodiments, vectors comprise non-coding sequences corresponding to at least one, preferably between one and twelve and/or four or more first and second Pax-responsive OP-1 modulating elements, respectively. First Pax-responsive sites correspond approximately to bases 108-121, 139-154, 157-167, 365-378, 497-511, 598-613, 1123-1140, 1144-1161, 1285-1297, 1750-1762, 2001-2023, 2365-2378 and 2931-2944 of SEQ. ID No. 1. Second Pax-responsive sites correspond approximately to bases 491-503, 737-747, 891-903, and 994-1006 of SEQ. ID No. 1.
In another aspect, the invention provides a cell comprising a reporter gene whose regulation is mediated by one or more of the Pax-responsive OP-1 non-coding sequences defined above. In one embodiment, the cell is transfected with a reporter gene in operative association with at least one Pax responsive site. In another embodiment, the present invention provides a cell comprising a transfected vector encoding a reporter gene operatively associated with at least two DNA sequences, the first comprising at least part of a sequence selected from SEQ. ID No. 2 while the second comprises at least part of a sequence selected from SEQ. ID No. 3, including allelic, species and other sequence variants of the foregoing. In yet another embodiment, cells of the present invention are co-transfected with expression vectors encoding Pax gene expression products such as, for example, Pax 2 and/or Pax 6.
In another aspect, the invention provides kits useful in the design and/or identification of OP-1 expression modulating compounds. As used herein a xe2x80x9ckitxe2x80x9d comprises a cell comprising a reporter gene in operative association with an OP-1 non-coding DNA sequence and the reagents necessary for detecting expression of the reporter gene. The portion of OP-1 noncoding DNA chosen can be any of the various sequences which have been described herein above.
Following this disclosure, medium flux screen assays, and kits therefore, for identifying modulators of morphogen expression, such as OP-1 expression, are available. These compounds can be naturally occurring molecules, or they can be designed and biosynthetically created using a rational drug design and an established structure/function analysis methodology. The compounds can be amino acid-based or can be composed in part or wholly of non-proteinaceous synthetic organic molecules.
The OP-1 expression modulating compounds thus identified can be produced in reasonable quantities, including commercially significant quantities, using standard recombinant expression or chemical synthesis technology well known and characterized in the art and/or as described herein. For example, automated means for the chemical synthesis of nucleic and amino acid sequences are commercially available. Alternatively, promising candidate compounds can be modified using standard biological or chemical methodologies to, for example, enhance the binding affinity of the compound for a DNA element and the preferred candidate compound derivative then can be produced in quantity.
Once a candidate compound has been identified and produced, it can be further tested for its effect on OP-1 expression. For example, a compound which upregulates (increases) the production of OP-1 (e.g., an OP-1 agonist) in a kidney cell line is a candidate for systemic administration. The candidate compound can be assayed in an animal model to determine the candidate molecule""s efficacy in vivo. For example, the ability of a compound to upregulate levels of circulating OP-1 in vivo can be used to correct bone metabolism diseases such as osteoporosis (See, for example, PCT/US92/07932, above). Conversely, compounds which down regulate (decrease) the production of OP-1 (e.g., OP-1 antagonists) are also contemplated to be useful. Useful in vivo animal models for systemic administration are disclosed in the art and below.
As is well known in the art, OP-1 is differentially expressed in different cell types. Accordingly, it is further anticipated that a candidate compound will have utility as an inducer of OP-1 expression in one cell type but not in another. Thus, the invention further contemplates testing a candidate compound for its utility in modulating expression of OP-1 in a tissue specific manner in vivo.
Thus, in view of this disclosure, one of ordinary skill in recombinant DNA and tissue culture techniques can design and construct appropriate DNA vectors and transfect cells with appropriate DNA sequences for use in the method according to the invention to assay for compounds which modulate the expression of OP-1. These identified compounds can be used to modulate OP-1 production and its effective concentrations in both in vivo and in vitro.